Dendritic cells (DCs) belong to the bone marrow-derived cell lineage, are present throughout the body in multiple tissues, and function as the central part of the mammalian immune system. Their main function is to process antigen material and present it on their surface to other cells of the immune system. Thus, dendritic cells function as antigen-presenting cells (APCs), and they do so more efficiently than any other type of APC, such as macrophages. DCs also act as messengers between innate and adaptive immunity, through a range of cell surface receptors that capture microbes and trigger information which is then transmitted to lymphocytes and cells of the innate immunesystem.
Dendritic cells are present in tissues that are in direct contact with the external environment, such as the skin (where there is a specialized dendritic cell type called Langerhans cells) and the inner lining of the nose, lungs, stomach and intestines. They can also be found in an immature state (iDCs) in the blood. Once activated, they migrate to the lymph nodes where they interact with T cells and B cells to initiate and shape the adaptive immune response. At certain development stages, they grow branched projections, the dendrites, that give the cell its name (déndron being Greek for “tree”). While similar in appearance, these are structures that can be distinguished from the dendrites of neurons.
Three types of DCs have been defined in human blood and these are the CD1C+ myeloid DCs, the CD141+ myeloid DCs and the CD303+ plasmacytoid DCs. This represents the nomenclature proposed by the IUIS nomenclature committee. In dendritic cells belonging to the myeloid lineage, the similar morphology results in a very large contact surface to their surroundings, compared to overall cell volume. Plasmacytoid DC have a more rounded shape, comparable to plasma cells.
Myeloid dendritic cells are made up of at least two subsets that can be distinguished by the reciprocal expression of CD141 (also known as BCDA3) and CD1C (also know as BDCA1). CD141-expressing DC represent the more common myeloid cells, often referred to as DC-1, and these cells represent major stimulator of the CD8 T cell response. The extremely rare myeloid CD1C expressing DC is often referred to as DC-2, and its function continues to be analyzed and defined. It has been reported to have a function in combating wound infection. Myeloid DC-1 cells express MHC class I and II molecules for antigen presentation, and a range of other molecules relevant for interacting with both the innate and the adaptive immune system. They can also secrete multiple cytokines, including IL-12, and express various kinds of Toll-like receptors (TLR) such as TLR2 and TLR4.
Plasmacytoid dendritic cells resemble plasma cells in appearance, but have certain functional characteristics similar to myeloid dendritic cells. They can produce high amounts of interferon-alpha and thus became known as IPC (interferon-producing cells) before their dendritic cell nature was revealed. Plasmacytoid dendritic cells express TLR7 and TLR9, and can be distinguished form myeloid DC by the expression of CD303, also known as BDCA-2.
Lymphoid and myeloid DCs evolve from lymphoid or myeloid precursors respectively and thus are both of hematopoietic origin. The blood DCs are typically identified and enumerated through antibodies that specifically bind to certain markers, and antibody-binding can be detected by flow cytometry through fluorescent and other markers attached to the antibodies.
Dendritic cells are derived from hematopoietic bone marrow progenitor cells, and these progenitor cells initially transform into immature dendritic cells (iDCs). These immature cells are characterized by high endocytic activity, in keeping with their efficient capture of antigens, and in this stage, their ability to activate T-cells is still poor. This coincides with low expression of co-stimulatory molecules and limited ability to secrete certain cytokines. Immature dendritic cells constantly sample the surrounding environment for pathogens such as viruses and bacteria. This is done through pattern recognition receptors (PRRs) such as the TLRs. TLRs recognize specific chemical signatures found on subsets of pathogens and tumour tissue. Immature dendritic cells may also phagocytose small quantities of membrane from live cells, in a process called nibbling.
Once they have come into contact with antigens presented by the environment (such as microbes or tumor cells), immature dendritic cells are triggered to differentiate into mature dendritic cells and begin to migrate to the lymph nodes. Immature dendritic cells phagocytose pathogens and degrade their proteins into small pieces, and upon maturation present those fragments at their cell surface using MHC molecules. Simultaneously, they up-regulate cell-surface receptors that act as co-receptors in T-cell activation such as CD83, CD40 and others, thus greatly enhancing their ability to activate T-cells. In addition, they up-regulate, a chemotactic receptor that induces the dendritic cell to travel through the blood stream to the spleen or through the lymphatic system to lymph nodes. Here they act as antigen-presenting cells: they activate helper T-cells and killer T-cells as well as B-cells by presenting them with antigens derived from pathogens or tumors, alongside non-antigen specific co-stimulatory signals.
Every T-cell is specific to one particular antigenic peptide presented in MHC class I or II molecules, through receptors that are clonally expressed and are termed T cell receptors (TCRs). Only dendritic cells, are able to activate resting naïve T-cells when the matching antigen-MHC complex is presented to their particular TCR. Other antigen presenting cell types, such as macrophages and B cells, do not have the ability to trigger native resting T cells, and can only activate memory T cell. Because dendritic cells can activate both memory and naive T cells, they are often refereed to as professional antigen-presenting cells, and they are the most potent of all the antigen-presenting cells.
Myeloid DC can be generated from monocytes, white blood cells which circulate in the body and, depending on the right signal, can turn into either dendritic cells or macrophages. The monocytes in turn are formed from stem cells in the bone marrow. Monocyte-derived dendritic cells can be generated in vitro from peripheral blood mononuclear cells (PBMCs). Plating of PBMCs in a tissue culture flask permits adherence of monocytes. Treatment of these monocytes with interleukin 4 (IL-4) and granulocyte-macrophage colony stimulating factor (GM-CSF) leads to differentiation to immature dendritic cells (iDCs) in about a week. Subsequent treatment with tumor necrosis factor (TNF) further differentiates the iDCs into mature dendritic cells.
Dendritic cells are constantly in communication with other cells in the body. This communication can take the form of direct cell-to-cell contact based on the interaction of cell-surface proteins. An example of this includes the interaction of the membrane proteins of the B7 family of dendritic cells, CD80 (B7.1) and CD86 (B7.2), with CD28 and CTLA4 on T cells. In addition, cellular communication of DC with their environment takes place over a distance via cytokines. For example, stimulating dendritic cells in vitro with microbial extracts causes the dendritic cells to rapidly begin producing IL-12. IL-12 is a signal that helps send naive CD4 T cells towards a Th1 phenotype. The ultimate consequence is priming and activation of the immune system for attack against the antigens which the dendritic cell presents on its surface. However, there are differences in the cytokines produced, depending on the type of dendritic cell. The plasmacytoid DC has the ability to produce large amounts of type-1 IFN's, which recruit more activated macrophages to allow phagocytosis.
Given their unique role in initiating primary immune responses, vaccine products prepared from patient-derived cultured DC are under development in a broad range of permutations (Galluzi et al., 2012). Such products can be prepared from peripheral blood or from bone marrow, and form part of the vaccine arsenal that is being evaluated in cancer patients (Palucka et al., 2011). Such DC based vaccines have shown to be safe and well-tolerated, and they do induce antigen-specific CD4+ and CD8+ effector T-cells responses in some patients. Nevertheless, efficacy of such products has been documented in only limited patient numbers, and they have not generated consistent clinical success. This is mainly attributable to the fact that the potency of such patient-based products is impossible to standardize. It is feasible to develop standard operating procedures for harvesting dendritic cell precursors from blood or bone marrow and for subsequent generation functional dendritic cells, but patient-to-patient variability with respect to functional differences will always preclude full reproducibility. Furthermore, there are substantial hurdles for large scale implementation of such patient-based products, since production is laborious, time-consuming and therefore expensive. In addition, there are indications for an inferior T cell-stimulatory phenotype of DC derived from advanced cancer patients, which would also argue against the use of autologous DC. Novel approaches for dendritic cell vaccines are therefore urgently needed.
One way of overcoming these drawbacks would be to generate DC products from sustainable cell lines that could be applied as allogeneic products. This would bypass the need to harvest dendritic cells from individual patients, and allow the production of precisely characterized, predictable and consistent dendritic cell products. In order to qualify for a commercial scale manufacturing process, such a cell line would need to have the following characteristics. First of all the cell line should be clonal and easy to maintain in culture. It also should have reproducible population doubling times, and consistent behavior with respect to responsiveness to differentiation and maturation signals to generate mature functional DC.
A number of cell lines have been explored for this purpose, including KG-1, THP-1, HL-60 and MUTZ-3 (Larsson K., et al., 2006 van Helden et al., 2008 and references therein). From these cell lines, only MUTZ-3-derived DCs closely resemble primary DCs prepared from blood (Larsson et al., 2006 van Helden et al., 2008, Santegoets et al., 2006a, 2006b, and Masterson et al., 2002). Notwithstanding the fact that these cell lines recapitulate some of the antigen presentation and other immunological features of dendritic cells, their limitations in biological and reproducible behavior prohibit their application as the basis for DC vaccines.
There remains a need in the art for cells and cell lines that may be employed in an industrial setting for the production of effective dendritic cells.